Minuet in D Major, K73h (K94)

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Overexpression of Caspase-8 induces apoptosis in an manner that is dependent upon the cell surface marker CD Yuan, , Current Opin. Other exemplary analytes are proteases that are exquisitely specific for human IgA of the lgA1 subtype. For general reviews of the IgA family of proteases, the reader is referred to Kornfeld et al, , Rev.

IgA protease production can be used to distinguish pathogenic from harmless Niesseriaceae: Mulks et al, , New Engl. Accordingly, assays for IgA protease production are potentially important in detecting and diagnosing microbial infection. Enzyme components of this invention contain a cross-link between amino acid residues. The term "cross-link" as used in this disclosure refers to the covalent chemical attachment of a cross-linking agent between two reactive amino acid residues in one or more polypeptide components of an active enzyme.

The link may, for example, be in the form of a disulfide or peptide bond between amino acid side chains, or formed as a result of contacting the polypeptide with a multifunctional cross-linking agent. Where the cross-link is intramolecular, a "cyclic" enzyme polypeptide is created. Cross-linked components of the invention comprise sequences that contain reactive amino acid residues that permit attachment of a cross-linking agent which comprises cross-linking moieties.

Other reactive groups found on amino acids include double bonds, alcohol groups, and the like. The cross-linking moiety is an integral part of the cross-linking agent and comprises a chemical moiety or functional group that enables the cross-linking agent to covalently bond to reactive amino acid residues. For example, a cross-linking agent useful to covalently couple thiol groups of proteins and peptides is bis-maleimidohexane BMH.

This cross-linking agent comprises a hexamethylene moiety having maleimido cross-linking moieties attached to each end of the hexamethylene.

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Wong, CRC Press, The amine groups of reactive amino acids in the enzyme component polypeptide may be cross-linked by reaction with an amino group-reactive moiety of the cross-linking agent. N-hydroxysuccinimide, dimethylsuberimidate, phenyldiisocyanate, phenyldiisothiocyanate, difluorodinitrobenzene and cyanic chloride are exemplary amino group reactive moieties suitable for use in cross-linking agents.

The thiol groups of reactive amino acids may be cross-linked by reaction with a sulfhydryl-reactive moiety of the cross-linking agent. Exemplary reactive groups are S-pyridyl, maleimide and bromoacetyl moieties.

The carboxyl groups of reactive amino acids may be cross-linked by reaction with carbodiimide or hydrazide moieties. The cross-linking moieties may be homo- or heterobifunctional, such that cross-linking between the appropriate residues, preferably at or near the N-terminal and C-terminal residues of the enzyme component, is accomplished. Thus, the cross-linking agent will have two reactive groups capable of covalent chemical attachment to the amino, thiol, carboxyl or aromatic groups of the desired amino acid residues of the enzyme component polypeptide.

When the polypeptide is formed by peptide synthesis, non-encoded amino acids may be inserted with activatable side-chains at particular points in the sequence. Upon activation, these residues will either link to an agent or to another point in the polypeptide chain. One example is the amino acid analog p-benzoyl-L-phenylalanine, the synthesis of which is described by Cauer et al.

If the desired analyte is a specific nucleotide sequence, i. The complementary sequence is usually between 4 and nucleotides in length, and is more typically about 20 to 40 nucleotides in length. The presence of the analyte nucleotide sequence results in hybridization between the single-stranded oligonucleotide sequence and the target nucleotide sequence. This newly formed duplex can then be cleaved by the addition of a double-stranded specific nuclease for example a restriction endonuclease or RNAase H , resulting in the linearization of cross-linked enzyme components that have formed duplexes with complementary analyte nucleic acid sequences, permitting assembly of the reagent enzyme and production of an assay signal.

Exemplary analytes include the nucleic acid sequences of infectious pathogens such as bacteria and viruses, including, for example, mycobacteria tuberculosis, streptococcus, N. Several additional groups of analytes or chemical environmental conditions can also be detected or measured using this technology.

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For example, the cross-linking agent selected could be one that is sensitive to cleavage by a specific chemical or environmental condition pH, temperature, oxidation, reduction, etc. Enzymes which do not directly cleave the cross-linking agent but whose activity results in the production of a metastable linker which can subsequently undergo cleavage indirect linker cleavage , are also detectable by this method. Such cross-linkers have the formula W CH 2 n -X-CH OY - CH 2 n -Z wherein W and Z are each a functional group selected from the group consisting of maleimide, succinimide and thiocyanate; n is a number from 1 to 10; X is oxygen, sulfur or nitrogen; and Y is an enzymaticaliy cleavable moiety selected from the group consisting of galactose, mannose, glucose, phosphate, butyrate and acetate.

In embodiment where the analyte to be measured is an enzyme, the cross-linking agent may comprise a chemical moiety that acts as a substrate site for the analyte enzyme. The enzyme reacts with the substrate site to destabilize the cross-linking agent, causing it to spontaneously hydrolyze to yield linearized enzyme component.

For example, acetal glycosides of aldehydes are known that, upon cleavage by a specific glycosidase enzyme, produce hydroxyacetals which spontaneously hydrolyze in aqueous solution to yield parent aldehydes. Based on this knowledge, a novel homobifunctional cross-linking agent was designed that contains a glycosyl acetal moiety.

Upon removal of the glycosyl residue by the action of the glycosidase enzyme, the hydroxy acetal is generated which spontaneously hydrolyses. The net result is cleavage within the cross-linking agent and consequent linearization of the cross-linked enzyme component.

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The rate in absorbance at nm was measured per minute between 4 and 6 minutes. The carboxyl groups of reactive amino acids may be cross-linked by reaction with carbodiimide or hydrazide moieties. A bactericide, such as sodium azide, can be present to prevent bacterial growth, especially during storage. Please obey the copyright laws of your country. Viral proteases such as HIV-1 and HIV-2 protease, coxsackie virus protease and herpes virus protease recognize specific peptide substrate sequences of the host's cellular proteins. For example, acetal glycosides of aldehydes are known that, upon cleavage by a specific glycosidase enzyme, produce hydroxyacetals which spontaneously hydrolyze in aqueous solution to yield parent aldehydes. This cross-linking agent comprises a hexamethylene moiety having maleimido cross-linking moieties attached to each end of the hexamethylene.

These glycosyl-containing cross-linking agents have the formula. Additionally, it is possible to construct cross-linking agents which are susceptible to cleavage by chemical or environmental conditions. For example, it is possible to select or design cross-linking agents having spacers between the cross-linking moieties which are cleaved by acid, base, oxidation, reduction, temperature, light and so forth.

An exemplary cross-linking agent useful for this purpose is the acid-labile 2,2-bis-maleimidoethoxypropane BMEP , which is cleaved by mild acid hydrolysis. Other such reagents are known in the art and may be employed in an analogous manner. The development of a cross-linked reassembling enzyme component from a prototype enzyme involves the steps of: The term "inserting" refers to design of the structure of the component wherein the inserted component is not found in exactly the same context in nature; no implication is intended as to the method of manufacture of the component.

Any arrangement of cross-link and heterologous sequence that prevents reassembly before cleavage, but permits reassembly after cleavage is suitable, and may be determined empirically. A systematic approach to obtaining cross-linked reassembling enzyme components is to base the location of the cross-link and heterologous sequence upon known structural features of the prototype. The functional elements of prototype enzyme can be mapped onto their structure by other techniques known in the art, such as photoreactive cross-linking, characterization using a panel of monoclonal antibodies, and mutation analysis.

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Ideally, full structural information of the prototype active enzyme is known by way of its amino acid sequence, its crystal structure, and the location of active site for the substrate. A large number of suitable enzymes including many of those exemplified in the previous section have been characterized in detail by way of crystal structure. Sequence and coordinate data is available through the Protein Database of the U.

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National Institutes of Health. Advanced computer algorithms are available e.

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Once structural features are known, possible positions for the cross-link are designed that prevent enzymatic activity when in place. For example, the cross-link may be positioned so as to prevent access of the substrate to the catalytic site, or to prevent access of an enzyme co-factor to its binding site. More typically, the cross-link is positioned so as to prevent a portion of the enzyme from associating with a second portion within or between polypeptide chains so as to form the catalytic site.

Particularly suitable are portions that are involved in an internal hydrophobic or other non- covalently bonded interface between subunits or regions of the molecule. Thus, the presence of the cross-link prevents the interface from forming until cleavage occurs.

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The importance of subunit or fragment interfaces in assembling enzymatic activity is demonstrated in several types of experiments. Where the cross-link is not itself cleavable by the analyte, a heterologous sequence is positioned within the polypeptide so as to permit the cross-linked portion to move out of the way, allowing access of the substrate to the active site, or allowing the active site to form, for example, by formation of a subunit interface.

Most usually, an inserted heterologous proteolytic cleavage recognition site is incorporated into the enzyme sequence internally, between the two reactive amino acid residues used for the cross-linking reaction. In combination or as an alternative to these predictive modeling techniques, cross-linked enzyme components of this invention are identified or refined by empirical testing.

A complementing subunit or fragment of an enzyme is pared down to the minimum number of amino acids necessary to assemble active enzyme complex. This is often more convenient with the smaller of the subunits in the assembled complex, in part, because of the ease of chemical synthesis of shorter sequences. Once the functional core of the component is identified, reactive amino acids can be added onto one or both ends that permit cross-linking around or back into the core.

Once an inactive cross-linked form of the enzyme component has been successfully designed and obtained, a heterologous protein recognition sequence is optionally incorporated into the structure. A heterologous protease recognition sequence, if present, is conveniently inserted between the terminal reactive amino acid and the core, which helps ensure that cleavage by the analyte will open the cyclized component in a way that permits it to assemble into an enzyme complex.

This approach is exemplified by the cyclized enzyme donor polypeptides described in the example section. In principle, the approach for generating a cyclized enzyme component based on another prototype enzyme is the same. Another example of a cyclized enzyme component of this invention is based on the prototype enzyme ribonuclease. Bovine pancreatic ribonuclease Rnase-A is one of the best characterized RNAses, and consists of a single chain of amino acids 13, mol wt.